Low-dose acetylsalicylic acid (aspirin) is widely used in the treatment and prevention of vascular disease. Aspirin prevents platelet activation and aggregation, but is also known to have platelet-independent vasoprotective effects. Aspirin promotes endothelium- dependent vasorelaxation but mechanisms by which it does so are not completely understood. The principal hypothesis of this application is that reversible acetylation of lysine residues in endothelial nitric oxide synthase (eNOS) by low-dose aspirin stimulates eNOS activity, and promotes endothelium-dependent vascular relaxation. The novelty of this application lies in 1) determining the role of lysine acetylation of eNOS by low-dose aspirin as a post-translational modification that promotes eNOS enzymatic activity and thereby endothelial NO production, and 2) exploring the role of the endogenous lysine deacetylase, histone deacetylase-3 (HDAC3), in reversing aspirin- stimulated lysine acetylation of eNOS and thereby antagonizing aspirin-induced endothelial NO production. The significance of this proposal lies in 1) identifying a novel molecular mechanism through which cardiovascular doses of aspirin may have vasoprotective effects, and 2) identifying a potentially exploitable endogenous mechanism that antagonizes the effect of aspirin on the vasculature. Although low-dose aspirin is effective in the prevention and treatment of cardiovascular disease, a significant proportion of patients on aspirin experience atherothrombotic events, underscoring the importance of further understanding how aspirin functions in the vasculature. This application, by looking at lysine acetylation of eNOS as a new mechanism for the effect of low-dose aspirin on endothelial function, and by identifying an endogenous antagonist to aspirin in the endothelium, may pave the way for future strategies that better harness the therapeutic potential of this widely used pharmaceutical. PUBLIC HEALTH RELEVANCE: Aspirin is widely used for the treatment and prevention of heart disease and has many beneficial effects on blood vessels. This research will explore whether aspirin chemically modifies nitric oxide synthase, a protein in blood vessels that improves vessel function and inhibits blood clot formation.